Proteolytic processing of the astrovirus capsid

To further characterize the nature of proteolytic processing of the astrovi rus capsid, we infected Caco-2 cells with a high multiplicity of astrovirus without trypsin in the presence of 5 to 10% fetal calf serum. These infect ions were characterized by pulse-chase labeling with [S-35]methionine, elec tron microscopy, gel electrophoresis of purified viral particles, and analy sis of infectivity of such particles with and without added trypsin, Pulse- chase experiments showed that the astrovirus capsid protein was initially t ranslated as an approximately 87-kDa protein. The 87-kDa capsid protein was rapidly converted intracellularly to a 79-kDa form which was found in smal ler amounts in the cell supernatant. Purification by differential centrifug ation yielded particles that appeared quite similar to trypsin-grown astrov irus particles by negatively stained electron microscopy. These particles w ere antigenically distinct from trypsin-treated virions as demonstrated by their various reactions with monoclonal antibodies in a solid-phase immunoa ssay, The purified trypsin-free particles were mainly composed of the 79-kD a capsid protein which tvas found to have an amino terminus at residue 71 o f the entire open reading frame 2 (ORF2) product. The cleavage site was ide ntified in a highly conserved region of the astrovirus ORF2 product. These trypsin-free particles were minimally infectious in cultured Caco-2 cells b ut became highly infectious (10(5)-fold increase) after trypsin but not chy motrypsin treatment, This trypsin-enhanced infectivity correlated with conv ersion of the 79-kDa capsid protein to three smaller peptides of approximat ely 34 29, and 26 kDa.